Genomic and EST sequencing projects are providing a sequence
infrastructure that is changing how protein biochemistry is
practiced. Data produced by tandem mass spectrometers provides
independent pieces of data about the components of a mixture.
By applying tandem mass spectrometry to the analysis of protein
mixtures the identities of the components can be determined
by the appropriate application of software in conjunction with
sequence databases. The approach we have developed uses proteolytic
digestion of the protein complex followed by separation of the
resulting mixture of peptides using microcolumn liquid chromatography
interfaced to a tandem mass spectrometer. Data-dependent operation
of the tandem mass spectra allows the highly efficient acquisition
of tandem mass spectra. As peptide mixtures become more complex,
highly resolving separation techniques are required to separate
the peptide components for tandem mass spectrometry. We have
examined several strategies to separate complex peptide mixtures
using microcolumn LC/LC and SPE/CE in conjunction with tandem
mass spectrometry. This approach has been applied to the analysis
of large protein complexes and to total cellular proteins.